1. Field of the Invention
The present invention relates to a method for preparing a composition containing Bacillus subtilis WG6-14 and a method for applying the composition.
2. Description of Related Art
Bacillus subtilis is a gram positive bacterium widespread in soil and on plant surfaces. Bacillus subtilis is known to be strictly aerobic (or facultative anaerobic), produces robust stress-tolerant endospores and is peritrichously flagellated. Bacillus subtilis has been used in probiotic food, such as natto, food additives, feed additives, source of enzymes, seed-protecting formulations, as well as plant disease control agent for many years. The members of Bacillus subtilis group are generally regarded as safe (GRAS) for animals and humans. Many Bacillus subtilis strains have been reported to produce antibiotics greatly inhibitory to plant pathogenic bacteria or fungi. The antagonistic effects reported include that against Uromyces phaseoli, Ceratocystis ulmi, Eutypa lata, Monilinia fructicola, Fusarium oxysporum, Aspergillus niger, Verticillium dahliae, and Puccinia pelargonii-zonalis. 
Voluminous literatures regarding to mechanisms of plant disease control by Bacillus subtilis have indicated a collective effect of antibiotics production, space and nutrient competition, induced resistance and plant growth promotion. The antibiotics, extracellular hydrolase, NH3 and volatile hydrocarbons are among the common metabolites of the bacterium shown to be responsible for the observed growth inhibition of pathogens. B. subtilis was known to produce more than 66 kinds of antibiotics. Most of them are circular peptides that survive protease digestion and have a molecular weight about 270 to 4,500 Da. The often encountered examples include mycobaillin, subtilin, bacilysin, bacillomycin, fungistatin, bulbiformin, bacillin, subsporin, bacillocin, mycosubtilin, fungocin, iturin, neocidin, eumycin and zwettermicin. Antibiotics are produced at the stationary phase when endospore formation become prominent. The biosynthesis of antibiotics is dependent on the provision of nutrition and is known to be a function of stresses in the cultural environment. The determinative factors include the kinds and concentration of carbon/nitrogen sources provided, temperature, pH and culture stage. The members of Bacillus subtilis group are renowned for their extraordinary competitiveness as colonizer in nature habitat. They survive well in rhizosphere, foliar surface and as well in vascular bundle. A rhizosphere strain of B. subtilis survived well in intercellular space of stem, root and vascular bundle of spruce and thus provided protective effect against diseases. As regards to induced resistance, Podile and Prakash have demonstrated an enhanced phenylalanine ammonia lyase (PAL) and disease resistance of pea seedlings by seed treatment using Bacillus subtilis BS AF1. Likewise, soil drenching of tomato plants using FZB24® B. subtilis was shown effective in reducing infection of Phytophthora infestans and Botrytis cinerea on the foliar tissue. The application of FZB24® B. subtilis reduced the foliar infection of phytophthora infestans by more than 50%, whereas that of Botrytis cinerea by approximately 20%. The enhanced resistance expressed on foliar tissue indicated clearly the functioning of induced resistance by the root treatment applied.
As for plant growth promotion, Bacillus spp. are known to produce various extracellular hydrolases including proteases which digest proteins into small peptides. Members of B. subtilis/amyloliquefaciens were shown producing phytic acid favorable for freeing phosphate from soil; the facilitated plant absorption leading to enhanced growth was reported (Krebs et al., 1998). The plant growth promoting effect has been demonstrated among B. subtilis group members including B. amyloliquefaciens, B. polymyxa, B. pumilus and B. subtilis; and their PGPR (plant growth promoting rhizobacteria) characteristics was well accepted. For practical application, a field test performed in Wisconsin demonstrated a 13.4 to 13.9% yield increase of soybean by application of B. cereus UW85. Likewise, by seed treatment of antagonistic Bacilli, it was reported the promotion of seed germination, seedling emergence, and beneficial Rhizobium colonization. At the same time the infection of Rhizoctonia solani AG4 was inhibited and the promoted growth and yield was observed.
It was reported that volatile metabolites produced by Bacillus spp. may inhibit the growth of fungal pathogens including Rhizoctonia solani and Pythium ultimum; the synthesis of antifungal volatiles appeared to be promoted by the provision of D-glucose and peptones. Recently, it was demonstrated the production of certain complex volatiles from some growth promoting Bacillus spp. The complex volatiles enhanced the growth of Arabidopsis thaliana seedlings and showed characteristics of 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol.
Application of beneficial Bacillus subtilis group on agricultural production has attracted increasing attention worldwide. Listed in Table 1 are the major products commercially available and their recommended uses. The products listed are mostly from USA and Europe, and are primarily recommended for combating soil borne fungal plant disease. In Taiwan, Taiwan-Bau, manufactured by Kuan-Hwa Chemical Co., Ltd., is by now the only product available. The recommended use was for the control of powdery mildew disease.
TABLE 1Commercialized Bacillus subtilis group productsCommercialized products andmanufacturersRecommended applicationsKodiak (containing B. subtilis GB03),For seed coating treatment, often used together withmanufactured by Gustafson, USA.chemical fungicides.Controls disease caused by Rhizoctoniasolani, Fusarium spp., Alternaria spp., andAspergillus spp..YieldShield powder (containing B. pumillusFor seed coating treatment, usedGB34), manufactured bytogether with hopper box treatment forGustafson, USA.combatting soil borne fungal disease on soybeanroot.Serenade wettable powder (containingSpraying application for the control ofB. subtilis QST716), manufactured bypowdery mildew, downy mildew,AgraQuest, USA.Cercospora leaf spot, early blight, lateblight, brown rot and fire blight.Companion liquid (containing B. subtilisSpraying and drenching for controllingGB03, B. lichniformis and B. megaterium),the diseases caused by Rhizoctoniamanufactured by Growthspp., Pythium spp., Fusarium spp. andProducts, USA.Phytophthora spp.HiStick N/T powder (containing B. subtilisFor seed coating and soaking toMB1600), manufactured byprevent diseases caused by RhizoctoniaBecker Underwood in GB andspp. and Aspergillus spp..MicroBio Group in USA.Epic (containing B. subtilis),Preventing root disease on cotton,manufactured by MicroBio, Englandpeanuts and kidney bean.Bactophyt (containing B. subtilis),Preventing fungal and bacteria diseasesmanufactured by Novosibirsk, Russiaon cereal crops and vegetablesFZB 24 (containing B. subtilis),Preventing soil borne and seedlingmanufactured by Bayer AG, Germanydiseases on vegetable, potato andtobacco.Taiwa-Bau (containing B. subtilis),Control of powdery mildew diseases.made by Kwaun-Hau Chemicals,Taiwan
Copper and antibiotic are among fungicides routinely applied for the control of bacterial diseases for many years; the development among bacteria pathogens chemical resistance are common. The copper resistance of Xanthomonas citri (causal agent of citrus canker) is one of the renowned examples, and the overuse of copper caused further deleterious effect on the natural environment. The potential threat of copper on beneficial soil microflora has been reported, and excessive/improper use of copper has resulted in increased population of mites, the declined tree vigor and significant yield loss of citrus. For the reduction of chemical pesticides and fertilizers application, a microbial biopesticide with antagonistic (to pathogen) and resistance enhancement (to the host) characteristics was known to be one of the best alternatives. For the control of diseases caused by Xanthomonads, the development of a practically useful microbial fungicide remained to be explored.
The present invention discloses a novel microbial formulation and a method that prepares high concentration viable Bacillus subtilis WG6-14 endospores by liquid fermentation.